Composition for treating hyperlipidemia comprising oxyntomodulin derivative

ABSTRACT

The present invention relates to a composition for preventing or treating hyperlipidemia, fatty liver disease or arteriosclerosis, comprising an oxyntomodulin derivative as an active ingredient. The oxyntomodulin derivative has a high ability to activate GLP-1 receptor and glucagon receptor compared to native oxyntomodulin and has the effects of reducing the blood total cholesterol, low-density cholesterol and triglyceride levels that were increased by high-fat diet, and increasing high-density cholesterol levels and the high-density cholesterol/low-density cholesterol ratio. Thus, the oxyntomodulin derivative can be effectively used for the treatment of hyperlipidemia and related diseases.

TECHNICAL FIELD

The present invention relates to a composition for preventing ortreating hyperlipidemia, fatty liver disease or arteriosclerosis, whichcomprises an oxyntomodulin derivative as an active ingredient, and to amethod for treating hyperlipidemia, fatty liver disease orarteriosclerosis using the composition.

BACKGROUND ART

In recent years, in Korea, the intake of fats from foods has increaseddue to economic growth and the westernization of eating habits, andmetabolic diseases such as hyperlipidemia, diabetes, hypertension,arteriosclerosis and fatty liver disease, which are caused by a lack ofexercise, have increased.

Hyperlipidemia refers to a condition associated with elevated levels oflipids, such as free cholesterol, cholesterol esters, phospholipids andtriglycerides, in blood. Hyperlipidemia can appear in three forms: (1)hypercholesterolemia, (2) hypertriglyceridemia, and (3) combinedhyperlipidemia (hypercholesterolemia and hypertriglyceridemia).Hyperlipidemia is generally classified into primary hyperlipidemia andsecondary hyperlipidemia. Primary hyperlipidemia is generally caused bygenetic defects, whereas secondary hyperlipidemia is caused by variousdisease conditions, drugs and dietary habits. In addition,hyperlipidemia is also caused by a combination of the primary andsecondary causes of hyperlipidemia. As criteria for the diagnosis ofhyperlipidemia, a total cholesterol level of 220 mg/dl or higher and atriglyceride level of 150 mg/dl or higher are generally used.

There are various forms of cholesterol that naturally occur in mammals.Low-density (LDL) cholesterol is known to be harmful to health, and itis known that an increase in LDL cholesterol increases the risk of heartdisease (Assman et al., Am. J. Card, 1996). In addition, high-density(HDL) cholesterol is regarded as good cholesterol and is essential forhealth, because it prevents atherosclerosis or the like.

Although hyperlipidemia does not show specific symptoms by itself,excessive lipids in blood adhere to the blood vessel walls to reduce theblood vessel size and cause atherosclerosis by inflammatory reactions.For this reason, coronary heart disease, cerebrovascular disease,obstruction of peripheral blood vessels, etc., can occur (E. Falk etal., Circulation, 1995). In addition, excessive blood lipids areaccumulated in liver tissue, and thus can cause fatty liver disease. Thefatty liver refers to a condition in which the ratio of fats in theweight of the liver is more than 5%. The fatty liver can be caused notonly by excessive intake of fats, but also by intake of alcohol.

Current methods that are used to reduce blood lipid levels includedietary therapy, exercise therapy and drug therapy. However, dietarytherapy or excise therapy is difficult to strictly control and perform,and the therapeutic effect thereof is also limited.

Drugs for reducing lipid levels, developed to date, include bile acidbinding resin, cholesterol-lowering drugs such as HMG-CoA reductaseinhibitors important in cholesterol biosynthesis, triglyceride-loweringdrugs such as fibric acid derivatives and nicotinic acid, etc. However,these drugs were reported to have side effects such as hepatic toxicity,gastrointestinal disorder and carcinogenesis. Thus, there is an urgentneed for the development of drugs that can be used to treathyperlipidemia and related diseases (e.g., atherosclerosis and fattyliver disease) while having less side effects.

As a candidate for such drugs, oxyntomodulin has recently receivedattention. Oxyntomodulin is produced from pre-glucagon and is a peptidethat can bind to both glucagon-like peptide-1 (GLP-1) and glucagonreceptor to perform dual function. Because of such characteristics,oxyntomodulin has been studied for various purposes, including thetreatment of obesity, hyperlipidemia and fatty liver disease. However,oxyntomodulin has a problem in that it should be administered at a highdose, because it has a short half-life in vivo and the activity thereofis insufficient for use in the treatment of obesity, hyperlipidemia andfatty liver disease.

Accordingly, the present inventors have developed an oxyntomodulinderivative having increased activity compared to native oxyntomodulinand have found that the oxyntomodulin derivative reduced the content andratio of lipids in blood in a hyperlipidemia-induced hamster model,indicating that the derivative can be effectively used for the treatmentof hyperlipidemia diseases, thereby completing the present invention.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide a composition forpreventing or treating hyperlipidemia, fatty liver disease oratherosclerosis, which contains an oxyntomodulin derivative as an activeingredient.

Another object of the present invention is to provide a method fortreating hyperlipidemia, fatty liver disease or atherosclerosis, themethod comprising a step of administering an oxyntomodulin derivative toa subject.

Still another object of the present invention is to provide the use ofan oxyntomodulin derivative in the preparation of a medicament forpreventing or treating hyperlipidemia, fatty liver disease oratherosclerosis.

Solution to Problem

To achieve the above objects, in one aspect, the present inventionprovides a composition for preventing or treating hyperlipidemia, fattyliver disease or atherosclerosis, which contains an oxyntomodulinderivative as an active ingredient.

As used herein, the term “oxyntomodulin” refers to a peptide producedfrom pre-glucagon that a precursor of glucagon. In the presentinvention, oxyntomodulin is meant include native oxyntomodulin and itsprecursor, analog (derivative), fragment and variant. Preferably,oxyntomodulin has an amino acid sequence of SEQ ID NO: 1(HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).

As used herein, the term “oxyntomodulin variant” is a peptide that hasone or more amino acid residues different from those of the amino acidsequence of native oxyntomodulin and possesses a function of activatingGLP-1 and glucagon receptors. The oxyntomodulin variant can be preparedby any one of substitution, addition, deletion, modification, or acombination thereof of some amino acids of native oxyntomodulin.

As used herein, the term “oxyntomodulin derivative” refers to a peptide,peptide derivative or peptide mimic that is prepared by the addition,deletion or substitution of some amino acids of native oxyntomodulin andcan activate both GLP-1 receptor and glucagon receptor at a high levelcompared to the level activated by native oxyntomodulin.

As used herein, the term “oxyntomodulin fragment” refers to a fragmenthaving an addition or deletion of one or more amino acids at the aminoor carboxyl terminal end of native oxyntomodulin, in which the addedamino acids may also be non-naturally occurring amino acids (e.g.,D-type amino acid). Such amino acids have a function of regulating bloodglucose levels in vivo.

Methods for preparing the oxyntomodulin variant, derivative and fragmentmay be used alone or in combination. For example, the present inventionincludes a peptide that has one or more amino acids different from thoseof native peptide and deamination of the N-terminal amino acid residuesand has a function of activating both GLP-1 receptor and glucagonreceptor.

Amino acids mentioned herein are abbreviated according to thenomenclature rules of IUPAC-IUB as follows:

Alanine A; Arginine R;

Asparagine N; Aspartic acid D;

Cysteine C; Glutamic acid E;

Glutamine Q; Glycine G;

Histidine H; Isoleucine I;

Leucine L; Lysine K;

Methionine M; Phenylalanine F

Proline P; Serine S;

Threonine T; Tryptophan W;

Tyrosine Y; Valine V.

In the present invention, the oxyntomodulin derivative encompasses anypeptide that is prepared by the substitution, addition, deletion orpost-translational modification (e.g., methylation, acylation,ubiquitination, or intramolecular covalent bonding) of amino acids inthe amino acid sequence of SEQ ID NO: 1 and can activate both theglucagon and GLP-1 receptors. Upon substitution or addition of aminoacids, not only 20 amino acids commonly found in human proteins, butalso atypical or non-naturally occurring amino acids can be used.Commercial sources of atypical amino acids include Sigma-Aldrich,ChemPep Inc., and Genzyme Pharmaceuticals. The peptides including theseamino acids and atypical peptide sequences may be synthesized andpurchased from commercial suppliers, for example, American PeptideCompany or Bachem (USA) or Anygen (Korea).

In a specific embodiment of the present invention, the oxyntomodulinderivative of the present invention is a novel peptide including theamino acid sequence of the following formula 1:

R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2  [Formula1]

wherein

R1 is histidine, desamino-histidyl, dimethyl-histidyl(N-dimethyl-histidyl), beta-hydroxyimidazopropionyl, 4-imidazoacetyl,beta-carboxy imidazopropionyl or tyrosine;

X1 is Aib (aminosiobutyric acid), d-alanine, glycine,Sar(N-methylglycine), serine, or d-serine;

X2 is glutamic acid or glutamine;

X3 is leucine or tyrosine;

X4 is serine or alanine;

X5 is lysine or arginine;

X6 is glutamine or tyrosine;

X7 is leucine or methionine;

X8 is aspartic acid or glutamic acid;

X9 is glutamic acid, serine, alpha-methyl-glutamic acid or is deleted;

X10 is glutamine, glutamic acid, lysine, arginine or serine or isdeleted;

X11 is alanine, arginine or valine or is deleted;

X12 is alanine, arginine, serine or valine or is deleted;

X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or isdeleted;

X14 is aspartic acid, glutamic acid or leucine or is deleted;

X15 is phenylalanine or is deleted;

X16 is isoleucine or valine or is deleted;

X17 is alanine, cysteine, glutamic acid, lysine, glutamine oralpha-methyl-glutamic acid or is deleted;

X18 is tryptophan or is deleted;

X19 is alanine, isoleucine, leucine, serine or valine or is deleted;

X20 is alanine, lysine, methionine, glutamine or arginine or is deleted;

X21 is asparagine or is deleted;

X22 is alanine, glycine or threonine or is deleted;

X23 is cysteine or lysine or is deleted;

X24 is a peptide having 2 to 10 amino acids consisting of a combinationof alanine, glycine and serine or is deleted; and

R2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK(SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQID NO: 39), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40) or is deleted (excludedif the amino acid sequence of formula 1 is identical to that of SEQ IDNO: 1).

In order to increase the activity of wild-type oxyntomodulin for theglucagon receptor and the GLP-1 receptor, the oxyntomodulin derivativeof the present invention may be substituted with 4-imidazoacetylobtained by deletion of the alpha carbon of histidine at position 1 ofthe amino acid sequence of SEQ ID NO: 1, desamino-histidyl obtained bydeletion of the N-terminal amino group, dimethyl-histidyl(N-dimethyl-histidyl) obtained by modification of the N-terminal aminogroup with two methyl groups, beta-hydroxy imidazopropionyl obtained bysubstitution of the N-terminal amino group with a hydroxyl group, orbeta-carboxy imidazopropionyl obtained by substitution of the N-terminalamino group with a carboxyl group. In addition, the GLP-1receptor-binding region may be substituted with amino acids that enhancehydrophobic and ionic bonds or a combination thereof. A portion of theoxyntomodulin sequence may be substituted with the amino acid sequenceof GLP-1 or Exendin-4 to increase the activity of the GLP-1 receptor.

Further, a portion of the oxyntomodulin sequence may be substituted witha sequence that enhances alpha helix. Preferably, amino acids atpositions 10, 14, 16, 20, 24 and 28 of the amino acid sequence offormula 1 may be substituted with amino acids or amino acid derivativesconsisting of Tyr(4-Me), Phe, Phe(4-Me), Phe(4-C1), Phe(4-CN),Phe(4-NO₂), Phe(4-NH₂), Phg, Pal, Nal, Ala(2-thienyl) and Ala(benzothienyl) that are known to stabilize alpha helix, and the type andnumber of alpha helix-stabilizing amino acid or amino acid derivativesto be inserted are not limited.

Preferably, amino acids at positions 10 and 14, 12 and 16, 16 and 20, 20and 24, and 24 and 28 of the amino acid sequence may be also substitutedwith glutamic acid or lysine so as to form rings, and the number ofrings to be inserted is not limited. Most preferably, the oxyntomodulinderivative may have an amino acid sequence selected from among thefollowing formulas 1 to 6.

In a specific embodiment, the oxyntomodulin derivative of the presentinvention is a novel peptide including the amino acid sequence of thefollowing formula 2, obtained by substitution of the amino acid sequenceof oxyntomodulin with that of exendin or GLP-1:

R1-A-R3  [Formula 2]

In another specific embodiment, the oxyntomodulin derivative of thepresent invention is a novel peptide including the amino acid sequenceof the following formula 3, which is prepared by linking a portion ofthe amino acid sequence of oxyntomodulin and a portion of the amino acidsequence of exendin or GLP-1 via a proper amino acid linker:

R1-B-C-R4  [Formula 3]

In still another specific embodiment, the oxyntomodulin derivative ofthe present invention is a novel peptide including the amino acidsequence of the following formula 4, wherein a portion of the amino acidsequence of oxyntomodulin is substituted with an amino acid capable ofenhancing the binding affinity to GLP-1 receptor, for example, Leu atposition 26 which binds with GLP-1 receptor by hydrophobic interactionis substituted with the hydrophobic residue Ile or Val.

R1-SQGTFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N-D8-R3  [Formula 4]

In still another specific embodiment, the oxyntomodulin derivative ofthe present invention is a novel peptide including the amino acidsequence of the following formula 5, wherein a portion of the amino acidsequence of native oxyntomodulin is deleted, added, or substituted withother amino acids in order to increase the abilities of nativeoxyntomodulin to activate GLP-1 receptor and glucagon receptor:

R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5-FV-E6-WLMNT-E7-R5  [Formula 5]

In formulas 2 to 5, R1 is as described in formula 1;

A is selected from the group consisting of SQGTFTSDYSKYLDSRRAQD-FVQWLMNT(SEQ ID NO: 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42),SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43),GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO: 44),GQGTFTSDYS-RQMEEEAVRLFIEWLKNG (SEQ ID NO: 45),GEGTFTSDL-SRQMEEEAVRLFIEWAA (SEQ ID NO: 46), andSQGTFTSDYSRQMEEEAVRL-FIEWLMNG (SEQ ID NO: 47);

B is selected from the group consisting of SQGTFTSDYSKYLDSRRAQD-FVQWLMNT(SEQ ID NO: 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42),SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43),GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO: 44),GQGTFTSDYS-RQMEEEAVRLFIEWLKNG (SEQ ID NO: 45),GEGTFTSDL-SRQMEEEAVRLFIEWAA (SEQ ID NO: 46),SQGTFTSDYSRQMEEEAVRL-FIEWLMNG (SEQ ID NO: 47), GEGTFTSDLSRQMEEEAVRLFIEW(SEQ ID NO: 48), and SQGTFTSDYSRYLD (SEQ ID NO: 49);

C is a peptide having 2 to 10 amino acids consisting of a combination ofalanine, glycine and serine;

D1 is serine, glutamic acid or arginine;

D2 is arginine, glutamic acid or serine;

D3 is arginine, alanine or valine;

D4 is arginine, valine or serine;

D5 is glutamine, arginine or lysine;

D6 is isoleucine, valine or serine;

D7 is methionine, arginine or glutamine;

D8 is threonine, glycine or alanine;

E1 is serine, Aib, Sar, d-alanine or d-serine;

E2 is serine or glutamic acid;

E3 is arginine or lysine;

E4 is glutamine or lysine;

E5 is aspartic acid or glutamic acid;

E6 is glutamine, cysteine or lysine;

E7 is cysteine or lysine or is deleted;

R3 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) orGPSSGAPPPSK (SEQ ID NO: 37);

R4 is HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQ ID NO: 39)or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40); and

R5 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) orGPSSGAPPPSK (SEQ ID NO: 37) or is deleted (excluded if the amino acidsequences of formulas 2 to 5 are identical to that of SEQ ID NO: 1).

Preferably, the oxyntomodulin derivative of the present invention may bea novel peptide of the following formula 6:

R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2  [Formula6]

wherein R1 is histidine, desamino-histidyl, 4-imidazoacetyl or tyrosine;

X1 is Aib(aminosiobutyric acid), glycine, serine or d-serine;

X2 is glutamic acid or glutamine;

X3 is leucine or tyrosine;

X4 is serine or alanine;

X5 is lysine or arginine;

X6 is glutamine or tyrosine;

X7 is leucine or methionine;

X8 is aspartic acid or glutamic acid;

X9 is glutamic acid or alpha-methyl-glutamic acid or is deleted;

X10 is glutamine, glutamic acid, lysine or arginine or is deleted;

X11 is alanine or arginine or is deleted;

X12 is alanine or valine or is deleted;

X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or isdeleted;

X14 is aspartic acid, glutamic acid or leucine or is deleted;

X15 is phenylalanine or is deleted;

X16 is isoleucine or valine or is deleted;

X17 is alanine, cysteine, glutamic acid, glutamine oralpha-methyl-glutamic acid or is deleted;

X18 is tryptophan or is deleted;

X19 is alanine, isoleucine, leucine or valine or is deleted;

X20 is alanine, lysine, methionine or arginine or is deleted;

X21 is asparagine or is deleted;

X22 is threonine or is deleted;

X23 is cysteine, lysine or is deleted;

X24 is a peptide having 2 to 10 amino acids consisting of glycine or isdeleted; and

R2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK(SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQID NO: 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40) or is deleted(excluded if the amino acid sequence of formula 6 is identical to thatof SEQ ID NO: 1).

More preferably, the oxyntomodulin derivative of the present inventionmay be selected from the group consisting of the peptides of SEQ ID NOs:2 to 34. Even more preferably, the oxyntomodulin derivative of thepresent invention may be an oxyntomodulin derivative described in Table1 of Example 2-1.

In an example of the present invention, oxyntomodulin derivatives havingthe amino acid sequences of SEQ ID NOs: 2 to 34, respectively, wereprepared, and it was found that the oxyntomodulin derivatives showedexcellent GLP-1 receptor and glucagon receptor activities compared tonative oxyntomodulin (Example 2). In other words, it could be seen fromthe above results that the oxyntomodulin derivative of the presentinvention exhibited excellent therapeutic effects againsthyperlipidemia, fatty liver disease or atherosclerosis by activating theGLP-1 receptor and the glucagon receptor.

The oxyntomodulin derivatives of the present invention are present inthe form of conjugates comprising various polymer in order to improvethe therapeutic effect and in vivo half-life of the derivatives.

The conjugate of the present invention shows an increase in the durationof effects compared to native oxyntomodulin, and the long-actingconjugates include an oxyntomodulin prepared by the modification,substitution, addition or deletion of the amino acids of nativeoxyntomodulin, an oxyntomodulin conjugated to a biodegradable polymersuch as polyethylene glycol (PEG), an oxyntomodulin conjugated to apolysaccharide such as albumin, antibody, elastin, fibronectin or chitinor to a long-acting protein such as an immunoglobulin fragment, anoxyntomodulin conjugated to fatty acid having the ability of binding toalbumin in vivo, or an oxyntomodulin en-capsulated in biodegradablenanoparticles, and the type of long-acting conjugate that is used in thepresent invention is not limited.

Preferably, the conjugate is a conjugate wherein an oxyntomodulinderivative having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2 to 34 is linked to an immunoglobulin Fcregion via a non-peptidyl polymer.

The immunoglobulin Fc region is a biodegradable polypeptide that ismetabolized in vivo, and thus is safe for use as a carrier for a drug.The immunoglobulin Fc region has a low molecular weight compared to theentire immunoglobulin molecule, and thus is advantageous in terms of thepreparation, purification and yield of conjugates. In addition, becausethe amino acid sequence differs between antibodies, a Fab portionshowing high non-homogeneity, and thus the homogeneity of the materialcan be greatly increased and the possibility of inducing bloodantigenicity can also be reduced.

As used herein, the term “immunoglobulin Fc region” refers to a proteinthat contains the heavy-chain constant region 2 (CH2) and heavy-chainconstant region 3 (CH3) of an immunoglobulin, excluding the heavy-chainand light-chain variable regions, the heavy-chain constant region 1(CH1) and the light-chain constant region 1 (CL1) of the immunoglobulin.It may further include a hinge region at the heavy-chain constantregion. Also, the immunoglobulin Fc region of the present invention maybe an expanded Fc region including part or all of the heavy-chainconstant region 1 (CH1) and/or the light-chain constant region 1 (CL1),except for the heavy-chain and light-chain variable regions, as long asit has an effect that is substantially equal to or better than thenative protein. Also, the immunoglobulin Fc region may be a regionhaving a deletion of a portion of a relatively long amino acid sequencecorresponding to CH2 and/or CH3. Specifically, the immunoglobulin Fcregion of the present invention may comprise 1) a CH1 domain, a CH2domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain,3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) acombination of one or more domains and an immunoglobulin hinge region(or a portion of the hinge region), or 6) a dimer of each domain of theheavy-chain constant regions and the light-chain constant region.

The immunoglobulin Fc region of the present invention includes a nativeamino acid sequence, and a sequence derivative (mutant) thereof. As usedherein, the term “amino acid sequence derivative” refers to a sequencethat is different from the native amino acid sequence due to thedeletion, insertion, non-conservative or conservative substitution or acombination thereof of one or more amino acid residues of the nativeamino acid sequence. For example, in the case of an IgG Fc, amino acidresidues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331,which are known to be important in binding, may be used as suitablesites for modification.

In addition, other various derivatives are possible, including one thathas a deletion of a region capable of forming a disulfide bond, or adeletion of some amino acid residues at the N-terminal end of native Fcor an addition of a methionine residue at the N-terminal end of nativeFc. Further, to remove effector functions, a deletion may occur in acomplement-binding site, such as a C1q-binding site and an ADCC(antibody dependent cell mediated cytotoxicity) site. Techniques ofpreparing such sequence derivatives of the immunoglobulin Fc region aredisclosed in International Patent Publication Nos. WO 97/34631 and WO96/32478.

Amino acid exchanges in proteins and peptides, which do not generallyalter the activity of the proteins or peptides, are known in the art (H.Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Themost commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in bothdirections. In addition, the Fc region may, if necessary, be modified byphospho-rylation, sulfation, acrylation, glycosylation, methylation,farnesylation, acetylation, amidation, and the like.

The above-described Fc derivatives show biological activity identical tothat of the Fc region of the present invention or have increasedstructural stability against heat, pH, or the like.

In addition, this Fc region may be obtained from native forms isolatedfrom humans and other animals including cows, goats, pigs, mice,rabbits, hamsters, rats and guinea pigs, or may be recombinants orderivatives thereof, obtained from transformed animal cells ormicroorganisms. Herein, the Fc region may be obtained from a nativeimmunoglobulin by isolating a whole immunoglobulin from a living humanor animal body and treating it with proteinase. When the wholeimmunoglobulin is treated with papain, it is digested into Fab and Fcregions, and when the whole immunoglobulin is treated with pepsin, it isdigested into pF′c and F(ab)₂ fragments. Fc or pF′c can be isolatedusing size exclusion chromatography or the like. Preferably, ahuman-derived Fc region is a recombinant immunoglobulin Fc regionobtained from a microorganism.

In addition, the immunoglobulin Fc region of the present invention maybe in the form of having native sugar chains or increased or decreasedsugar chains compared to a native form, or may be in a deglycosylatedform. The increase, decrease or removal of the immunoglobulin Fc sugarchains may be achieved by conventional methods such as a chemicalmethod, an enzymatic method and a genetic engineering method using amicroorganism. The Fc region obtained by removal of sugar chains from Fcshows a significant decrease in binding affinity to the C1q part of thefirst complement component C1 and a decrease or loss inantibody-dependent cell-mediated cytotoxicity or complement-dependentcytotoxicity, and thus does not induce unnecessary immune responses invivo. In this regard, an immunoglobulin Fc region in a deglycosylated oraglycosylated form may be more suitable to the object of the presentinvention as a drug carrier.

As used herein, the term “deglycosylation” refers to enzymaticallyremoving sugar moieties from an Fc region, and the term “aglycosylation”refers to an unglycosylated Fc region produced in a prokaryote,preferably E. coli.

Meanwhile, the immunoglobulin Fc region may be derived from humans orother animals including cows, goats, pigs, mice, rabbits, hamsters, ratsand guinea pigs, and preferably from humans.

In addition, the immunoglobulin Fc region may be derived from IgG, IgA,IgD, IgE, IgM, or a combination or hybrid thereof. Preferably, it isderived from IgG or IgM, which are among the most abundant proteins inhuman blood, and most preferably from IgG, which is known to enhance thehalf-lives of ligand-binding proteins.

As used herein, the term “combination” means that polypeptides encodingsingle-chain immunoglobulin Fc regions of the same origin are linked toa single-chain polypeptide of a different origin to form a dimer ormultimer. Specifically, a dimer or multimer may be formed from two ormore fragments selected from the group consisting of IgG Fc, IgA Fc, IgMFc, IgD Fc, and IgE Fc fragments.

As used herein, the term “hybrid” means that sequences corresponding totwo or more immunoglobulin Fc fragments of different origins are presentin a single-chain immunoglobulin Fc region. In the present invention,various forms of hybrid are possible. In other words, a hybrid composedof 1 to 4 domains selected from the group consisting of the CH1, CH2,CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible,and it may include a hinge.

Meanwhile, IgG can also be sub-classified into IgG1, IgG2, IgG3 andIgG4, and in the present invention, a combination or hybrid of thesesubclasses is also possible. Preferably, IgG is the IgG2 ad IgG4subclass, and most preferably, it is the Fc region of IgG4 thatsubstantially lacks effector functions such as complement-dependentcytotoxicity (CDC).

In other words, the most preferred immunoglobulin Fc region that is usedas a drug carrier in the present invention is an Fc region derived fromhuman IgG4. A human-derived Fc region is more preferable than anon-human-derived Fc region, which may act as an antigen in the humanbody and cause undesirable immune responses such as the production of anew antibody against the antigen.

As used herein, the term “non-peptidyl polymer” refers to abiocompatible polymer including two or more repeating units linked toeach other by any covalent bond in place of a peptide bond. In thepresent invention, the non-peptidyl polymer may be interchangeably usedwith the non-peptidyl linker.

The non-peptidyl polymer that can be used in the present invention maybe selected from the group consisting of polyethylene glycol,polypropylene glycol, an ethylene glycol/propylene glycol copolymer,polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran,polyvinyl ethyl ether, biodegradable polymers such as PLA (poly(lacticacid)) and PLGA (polylactic-glycolic acid), lipid polymers, chitins,hyaluronic acid, and combinations thereof. Preferably, the non-peptidylpolymer is polyethylene glycol. In addition, derivatives thereof knownin the art and derivatives that may be easily prepared by a method knownin the art are included in the scope of the present invention.

The peptide linker that is used in a fusion protein obtained by aconventional inframe fusion method has drawbacks in that it is easilycleaved by proteinase in vivo, and thus a sufficient effect ofincreasing the serum half-life of the active drug by a carrier cannot beobtained as expected. However, in the present invention, the polymerhaving resistance to proteinase can be used to maintain the serumhalf-life of the peptide, similar to the carrier. Therefore, anynon-peptidyl polymer can be used without limitation in the presentinvention, as long as it is a polymer having the aforementionedfunction, that is, a polymer having resistance to proteinase in vivo.The non-peptidyl polymer has a molecular weight in the range of 1 to 100kDa, and preferably 1 to 20 kDa. The non-peptidyl polymer of the presentinvention, which is linked to the immunoglobulin Fc region, may be onekind of polymer or a combination of different polymers.

The non-peptidyl polymer that is used in the present invention may havea reactive group capable of binding to the immunoglobulin Fc region andthe protein drug. The reactive group at both ends of the non-peptidylpolymer is preferably selected from the group consisting of a reactivealdehyde group, a propionaldehyde group, a butyraldehyde group, amaleimide group and a succinimide derivative.

The succinimide derivative may be succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. Inparticular, when the non-peptidyl polymer has a reactive aldehyde groupat both ends thereof, non-specific reactions can be minimized, and aphysiologically active polypeptide and an immunoglobulin can beeffectively bound to one and the other end of the non-peptidyl polymer,respectively. A final product generated by reductive alkylation with analdehyde bond is much more stable than that linked by an amide bond. Thealdehyde reactive group selectively binds to an N-terminus at a low pHand can form a covalent bond with a lysine residue at a high pH such aspH 9.0.

The reactive groups at both ends of the non-peptidyl polymer may be thesame or different. For example, the non-peptidyl polymer may possess amaleimide group at one end, and an aldehyde group, a propionaldehydegroup or a butyraldehyde group at the other end. When a polyethyleneglycol having a reactive hydroxy group at both ends thereof is used asthe non-peptidyl polymer, the hydroxy group may be activated to variousreactive groups by known chemical reactions, or a polyethylene glycolhaving a commercially available modified reactive group may be used soas to prepare the long acting conjugate of the present invention.

The conjugate of the present invention may be one in which one end ofthe non-peptidyl polymer and the other are linked to an amine group or athiol group of the immunoglobulin Fc region and the oxyntomodulinderivative, respectively.

The non-peptidyl polymer of the present invention has a functional groupat both ends which can be linked to either an immunoglobulin Fc regionor a protein drug. The functional groups can be an aldehyde group, apropionaldehyde group, a butyraldehyde group, a maleimide group and asuccinimide derivative (i.e., succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate),but are not limited thereto.

The reactive groups at both ends of the linker that is the non-peptidylpolymer may be the same or different. For example, the non-peptidylpolymer may have a maleimide group at one end and an aldehyde group, apropionaldehyde group or a butyraldehyde group at the other end. Forexample, when the non-peptidyl polymer has a reactive aldehyde group atone end and a reactive maleimide group at the other end, non-specificreactions can be minimized, and a physiologically active polypeptide andan immunoglobulin can be effectively bound to both ends of thenon-peptidyl polymer. According to an embodiment of the presentinvention, a conjugate was synthesized by linking oxyntomodulin or itsderivative to the immunoglobulin Fc region via a covalent bond using thenon-peptidyl polymer PEG including a propionaldehyde group alone or botha maleimide group and an aldehyde group.

The pharmaceutical composition of the present invention can be used forthe prevention or treatment of hyperlipidemia, fatty liver disease oratherosclerosis.

As used herein, the term “prevention” refers to all actions that inhibitor delay the development of a target disease. As used herein, the term“prevention” means administering the oxyntomodulin derivative of thepresent invention to inhibit or delay the development of hyperlipidemia,fatty liver disease or atherosclerosis, which shows an increase in bloodtotal cholesterol and low-density cholesterol levels and a decrease inhigh-density cholesterol levels.

As used herein, the term “treatment” refers to all actions thatalleviate, ameliorate or relieve the symptoms of the disease developed.As used herein, the term “treatment” means administering theoxyntomodulin derivative of the present invention to alleviate,ameliorate or relieve hyperlipidemia, fatty liver disease oratherosclerosis, which shows an increase in blood total cholesterol andlow-density cholesterol levels and a decrease in high-densitycholesterol levels.

As used herein, the term “hyperlipidemia” refers to a conditionassociated with ab-normally elevated levels of lipids, such as freecholesterol, cholesterol esters, phospholipids and triglycerides, inblood. Although hyperlipidemia does not show specific symptoms byitself, excessive lipids in blood adhere to the blood vessel walls toreduce the blood vessel size and cause atherosclerosis by inflammatoryreactions. For this reason, coronary heart disease, cerebrovasculardisease, obstruction of peripheral blood vessels, etc., can occur.

Thus, the pharmaceutical composition of the present invention can beused for the treatment of not only hyperlipidemia, fatty liver diseaseor atherosclerosis, but also coronary heart disease, cerebrovasculardisease, or obstruction of peripheral blood vessels.

As used herein, the term “fatty liver disease” refers to a condition inwhich the ratio of fats in the weight of the liver is more than 5%. Inthe present invention, fatty liver diseases include non-alcoholic fattyliver disease (NAFLD), alcoholic fatty liver disease, nutritional fattyliver disease, starvation fatty liver disease, obesity fatty liverdisease, diabetic fatty liver disease or steatohepatitis. Thenon-alcoholic fatty liver disease is meant to include primary andsecondary non-alcoholic fatty liver disease, but may preferably be anon-alcoholic fatty liver disease resulting from primary hyperlipidemia,diabetes or obesity.

In addition, in the present invention, non-alcoholic fatty liver diseaseis meant to include simple steatosis, non-alcoholic steatohepatitis, andliver fibrosis and liver cirrhosis which result from the progression ofsuch diseases.

Atherosclerosis refers to a vascular disease in which atheroma is formedas a result of deposition of cholesterol in the endothelium of bloodvessels and proliferation of en-dothelial cells.

In an example of the present invention, a long-acting oxyntomodulinderivative conjugate was prepared by linking the oxyntomodulinderivative to the immunoglobulin Fc region by a covalent bond usingpolyethylene glycol, and the prepared conjugate was administered tohamster animal models having hyperlipidemia induced by intake ofhigh-fat diet. As a result, it was shown that the group administeredwith the long-acting oxyntomodulin derivative conjugate according to thepresent invention showed a significant decrease in blood triglyceridelevels (FIG. 1), a significant decrease in blood total cholesterollevels (FIG. 2), and a significant decrease in blood low-density (LDL)cholesterol levels, compared to the hyperlipidemia-induced animalmodels. In addition, it was observed that the group administered withthe long-acting oxyntomodulin derivative conjugate according to thepresent invention showed a significant increase in blood high-density(HDL) cholesterol levels (FIG. 4) and a significant increase in theblood HDL-cholesterol/LDL-cholesterol ratio (FIG. 5), compared to thehyperlipidemia-induced animal models.

Further, it could be seen that the long-acting oxyntomodulin derivativeconjugate according to the present invention showed a decrease in bloodtotal cholesterol levels (FIG. 6) and a decrease in bloodLDL-cholesterol and triglyceride levels (FIG. 7), compared to VICTOZA®that is a commercial long-acting GLP-1 analog. In addition, it could beseen that administration of the long-acting oxyntomodulin derivativeconjugate of the present invention showed increases in bloodHDL-cholesterol level and the HDL/LDL-cholesterol ratio compared toadministration of VICTOZA®(FIGS. 8 and 9). Particularly, a long-actingconjugate of the peptide of SEQ ID NO: 25 with Fc showed significantincreases in blood HDL levels and HDL/LDL-cholesterol ratio compared toVICTOZA®.

In other words, the oxyntomodulin derivative according to the presentinvention reduce blood lipid levels, and thus can be used as an agentfor treating hyperlipidemia, fatty liver disease or arteriosclerosis. Inaddition, the conjugate of the present invention has an excellentability to activate GLP-1 receptor and glucagon receptor compared tonative oxyntomodulin and shows an increased blood half-life in vivo, andthus the activity thereof can be maintained in vivo for an extendedperiod of time.

The oxyntomodulin derivative of the present invention can increase theactivity of a factor (Protein kinase C-ζ or PKC-ζ) regulating theactivity of enzymes that are involved in the lipolysis of fats, andincrease the expression of a factor (Glut2) that is involved in thelipolysis of fats, thereby treating hyperlipidemia, fatty liver diseaseor arteriosclerosis, but the scope of the present invention is notlimited to the above mechanism of action.

The pharmaceutical composition of the present invention may furthercomprise a pharmaceutical agent exhibiting preventive or therapeuticeffects against hyperlipidemia, fatty liver disease or arteriosclerosis.Specifically, the composition of the present invention may furthercomprise a pharmaceutical agent known as an agent for treatinghyperlipidemia, fatty liver disease or arteriosclerosis in order toadminister the pharmaceutical agent in combination with the derivativeof the present invention.

Thus, the composition of the present invention may be administered aloneor in combination with other drugs in order to prevent or treathyperlipidemia, fatty liver disease or arteriosclerosis.

As used herein, the term “administration” means introducing a givenmaterial into a patient by any appropriate method. The derivative of thepresent invention may be administered by any general route, as long asit can reach a target tissue. Specifically, the derivative of thepresent invention may be administered intraperitoneally, intra-venously,intramuscularly, subcutaneously, intradermally, orally, locally,intranasally, intrapulmonarily or intrarectally, but is not limitedthereto. However, because the peptide is digested when beingadministered orally, the oral composition is preferably formulated sothat the active ingredient is coated or protected from degradation inthe stomach. Preferably, the composition of the present invention may beadministered in an injectable form. In addition, the pharmaceuticalcomposition of the present invention may be administered using anysystem capable of delivering the active ingredient to target cells.

The pharmaceutical composition comprising the oxyntomodulin derivativeof the present invention may further comprise a pharmaceuticallyacceptable carrier. For oral administration, pharmaceutically acceptablecarriers include a binder, a lubricant, a disintegrant, an excipient, asolubilizer, a dispersing agent, a stabilizer, a suspending agent, acolorant, and a flavorant. For injectable preparations, pharmaceuticallyacceptable carriers include a buffer, a preservative, an analgesic, asolubilizer, an isotonic agent, and a stabilizer. For topicaladministration, pharmaceutically acceptable carriers include a base, anexcipient, a lubricant, and a preservative.

The pharmaceutical composition of the present invention may beformulated in various dosage forms using the aforementionedpharmaceutically acceptable carriers. For example, for oraladministration, the pharmaceutical composition may be formulated intotablets, troches, capsules, elixirs, suspensions, syrups, wafers or thelike. For injectable preparations, the pharmaceutical composition may beprovided in the form of a unit dosage ampoule or a multiple dosagecontainer. The pharmaceutical composition may also be formulated intosolutions, suspensions, tablets, pills, capsules and sustained-releasepreparations.

Meanwhile, examples of the carrier, excipient and diluent suitable forformulation include lactose, dextrose, sucrose, sorbitol, mannitol,xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin,calcium phosphate, calcium silicate, cellulose, methylcellulose,microcrystalline cellulose, polyvinylpyrrolidone, water,methylhy-droxybenzoate, propylhydroxybenzoate, talc, magnesium stearateand mineral oils. In addition, the pharmaceutical composition of thepresent invention may further include fillers, anti-coagulating agents,lubricants, wetting agents, flavors, preservative and the like.

The dose of the pharmaceutical composition of the present invention isdetermined according to the kind of active ingredient, together withvarious factors such as the disease to be treated, the route ofadministration, the patient's age, sex and weight, and the severity ofthe disease.

The pharmaceutical composition of the present invention has a long invivo half-life and excellent-potency, and thus the number and frequencyof administration of the pharmaceutical composition can be significantlyreduced.

In another embodiment, the present invention provides a method fortreating hyperlipidemia, fatty liver disease or arteriosclerosis, themethod comprising a step of administering the oxyntomodulin derivativeof the present invention to a subject.

The above oxyntomodulin, hyperlipidemia, fatty liver disease andarteriosclerosis are as described above.

As used herein, the term “subject” refers to a subject suspected ofhaving hyperlipidemia, fatty liver disease or arteriosclerosis.Specifically, the term means mammals, including humans, rats anddomestic animals, which have or are at the risk of de-veloping the abovedisease. In addition, the subject may be any subject that can be treatedby the oxyntomodulin derivative of the present invention.

The therapeutic method of the present invention may compriseadministering a pharmaceutically effective amount of the pharmaceuticalcomposition comprising the conjugate. The total daily dose of thecomposition can be determined through appropriate medical judgment by aphysician, and the composition may be administered once or severaltimes. However, in view of the purpose of the present invention, thespecific therapeutically effective dose of the composition for anyparticular patient may vary depending on various factors well known inthe medical field, including the kind and degree of response to beachieved, concrete compositions according to whether other agents areused therewith or not, the patient′age, body weight, health condition,sex and diet, the time and route of administration, the secretion rateof the composition, the duration of treatment, other drugs used incombination or coincident with the composition of the present invention,and other factors known in the medical field.

In still another aspect, the present invention provides a method forpreparing an oxyntomodulin derivative conjugate.

The preparation method may comprise the steps of: (1) covalently linkinga non-peptidyl polymer having a reactive aldehyde, maleimide orsuccinimide group to the amine or thiol group of an oxyntomodulinderivative peptide; (2) separating the oxyntomodulin derivative peptide,having the non-peptidyl polymer covalently bonded thereto at positionsother than the amino terminal end, from the reaction mixture of step(1); and (3) covalently an immunoglobulin Fc region to the other end ofthe linked non-peptidyl polymer, thereby producing a peptide conjugatecomprising the immunoglobulin Fc region and the oxyntomodulin derivativepeptide, linked to one and the other end of the non-peptidyl polymer,respectively.

More specifically, the preparation method may comprise the steps of: 1)covalently linking a non-peptidyl polymer, having a reactive aldehydegroup and a reactive maleimide group at one and the other end thereof,respectively, to the cysteine residue of an oxyntomodulin derivative;(2) separating the oxyntomodulin derivative, having the non-peptidylpolymer covalently linked to the cysteine residue, from the reactionmixture of step (1); and (3) covalently an immunoglobulin Fc region tothe other end of the linked non-peptidyl polymer, thereby producing apeptide conjugate comprising the immunoglobulin Fc region and theoxyntomodulin derivative peptide, linked to one and the other end of thenon-peptidyl polymer, respectively.

In still another aspect, the present invention provides the use of theoxyntomodulin derivative in the preparation of a medicament forpreventing or treating hyperlipidemia, fatty liver disease orarteriosclerosis.

Advantageous Effects of Invention

The oxyntomodulin derivative of the present invention has a high abilityto activate GLP-1 receptor and glucagon receptor compared to nativeoxyntomodulin and exhibits the effects of reducing the blood totalcholesterol, low-density cholesterol and triglyceride levels that wereincreased by high-fat diet, and increasing high-density cholesterollevels and the high-density cholesterol/low-density cholesterol ratio.Thus, the oxyntomodulin derivative of the present invention can beeffectively used for the treatment of hyperlipidemia and relateddiseases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the change in blood triglyceride levels causedby administration of a long-acting oxyntomodulin derivative to high-fatdiet-induced hyperlipidemia hamsters (#: indicates a significantincrease compared to a general diet group within a confidence of 99.9%(p<0.001);*: indicates a significant decrease compared to a high-fatdiet group within a confidence of 99.9% (p<0.001).

FIG. 2 is a graph showing the change in blood total cholesterol levelscaused by administration of a long-acting oxyntomodulin derivative tohigh-fat diet-induced hyperlipidemia hamsters (#: indicates asignificant increase compared to a general diet group within aconfidence of 99.9% (p<0.001); *: indicates a significant decreasecompared to a high-fat diet group within a confidence of 99.9%(p<0.001).

FIG. 3 is a graph showing the change in blood LDL-cholesterol levelscaused by administration of a long-acting oxyntomodulin derivative tohigh-fat diet-induced hyperlipidemia hamsters (#: indicates asignificant increase compared to a general diet group within aconfidence of 99.9% (p<0.001); *: indicates a significant decreasecompared to a high-fat diet group within a confidence of 99.9%(p<0.001).

FIG. 4 is a graph showing the change in blood HDL-cholesterol levelscaused by administration of a long-acting oxyntomodulin derivative tohigh-fat diet-induced hyperlipidemia hamsters (*: indicates asignificant decrease compared to a high-fat diet group within aconfidence of 99% (p<0.01).

FIG. 5 is a graph showing the change in blood HDL/LDL-cholesterol levelscaused by administration of a long-acting oxyntomodulin derivative tohigh-fat diet-induced hyperlipidemia hamsters (*: shows a significantdecrease compared to a high-fat diet group within a confidence of 95%(p<0.05).

FIG. 6 is a graph showing the change in blood total cholesterol levelscaused by administration of VICTOZA® or a long-acting oxyntomodulinderivative to high-fat diet-induced hyperlipidemia hamsters (***:indicates a significant decrease compared to a high-fat diet groupwithin a confidence of 99.9% (p<0.001).

FIG. 7 is a graph showing the change in blood LDL-cholesterol levelscaused by administration of VICTOZA® or a long-acting oxyntomodulinderivative to high-fat diet-induced hyperlipidemia hamsters (***:indicates a significant decrease compared to a high-fat diet groupwithin a confidence of 99.9% (p<0.001).

FIG. 8 is a graph showing the change in blood HDL-cholesterol levelscaused by administration of VICTOZA® or a long-acting oxyntomodulinderivative to high-fat diet-induced hyperlipidemia hamsters (*:indicates a significant decrease compared to a high-fat diet groupwithin a confidence of 95% (p<0.05).

FIG. 9 is a graph showing the change in blood HDL/LDL-cholesterol levelscaused by administration of VICTOZA® or a long-acting oxyntomodulinderivative to high-fat diet-induced hyperlipidemia hamsters (**:indicates a significant decrease compared to a high-fat diet groupwithin a confidence of 99% (p<0.01).

FIG. 10 is a graph showing the change in blood triglyceride levelscaused by administration of VICTOZA® or a long-acting oxyntomodulinderivative to high-fat diet-induced hyperlipidemia hamsters (***:indicates a significant decrease compared to a high-fat diet groupwithin a confidence of 99.9% (p<0.001).

MODE FOR THE INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not intended tolimit the scope of the present invention.

Example 1 Production for Cell Line for In Vitro Activation Example 1-1Production of Cell Line Showing cAMP Response to GLP-1

Using a portion corresponding to the ORF (open reading frame) of cDNA(OriGene Technologies, Inc. USA) of the human GLP-1 receptor gene as atemplate, PCR was performed using reverse and forward primers includinga HindIII cleavage site and an EcoRI cleavage site, respectively,thereby obtaining a PCR product.

Forward primer: (SEQ ID NO: 50) 5′-CCCGGCCCCCGCGGCCGCTATTCGAAATAC-3′Reverse primer: (SEQ ID NO: 51) 5′-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3′

The PCR product was cloned into the known animal cell expression vectorx0GC/dhfr, thereby constructing the recombinant vector x0GC/GLP-1R.

The recombinant vector x0GC/GLP-1R was introduced into a CHO DG44 cellline, cultured in DMEM/F12 (10% FBS) medium, using lipofectamine(Invitrogene, USA), to obtain a transformant. The transformant wasincubated in a selective medium containing 1 mg/mL G418 and 10 nMmethotraxate, and monoclonal cell lines were selected therefrom. Then, acell line showing a good concentration-dependent cAMP response to GLP-1was finally selected from the monoclonal cell lines.

Example 1-2 Production of Cell Line Showing cAMP Response to Glucagon

Using a portion corresponding to the ORF (open reading frame) of cDNA(OriGene Technologies, Inc. USA) of the human glucagon receptor gene asa template, PCR was performed using reverse and forward primersincluding an EcoRI cleavage site and a XhoI cleavage site, respectively,thereby obtaining a PCR product.

Forward primer: (SEQ ID NO: 52) 5′-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3′Reverse Primer: (SEQ ID NO: 53) 5′-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3′

The PCR product was cloned into the known animal cell expression vectorx0GC/dhfr, thereby constructing the recombinant vector x0GC/GCGR.

The recombinant vector x0GC/GCGR was introduced into a CHO DG44 cellline, cultured in DMEM/F12 (10% FBS) medium, using lipofectamine(Invitrogene, USA), to obtain a transformant. The transformant wasincubated in a selective medium containing 1 mg/mL G418 and 10 nMmethotraxate, and monoclonal cell lines were selected therefrom. Then, acell line showing a good concentration-dependent cAMP response toglucagon was finally selected from the monoclonal cell lines.

Example 2 In Vitro Activity of Oxyntomodulin Derivatives Example 2-1Synthesis of Oxyntomodulin Derivatives

In order to measure the in vitro activities of oxyntomodulinderivatives, oxyntomodulin derivatives having the amino acid sequencesshown in Table 1 below.

Table 1

TABLE 1 Oxyntomodulin and oxyntomodulin derivatives SEQ ID NOs SequencesSEQ ID NO: 1 HSQGTFTSDYSKYLDSRRAQDFV QWLMNTKRNRNNIA SEQ ID NO: 2CA-SQGTFTSDYSKYLDEEAVRL FIEWLMNTKRNRNNIA SEQ ID NO: 3CA-SQGTFTSDYSKYLDERRAQD FVAWLKNTGPSSGAPPPS SEQ ID NO: 4CA-GQGTFTSDYSRYLEEEAVRL FIEWLKNGGPSSGAPPPS SEQ ID NO: 5CA-GQGTFTSDYSRQMEEEAVRL FIEWLKNGGPSSGAPPPS SEQ ID NO: 6CA-GEGTFTSDLSRQMEEEAVRL FIEWAAHSQGTFTSDYSKYLD SEQ ID NO: 7CA-SQGTFTSDYSRYLDEEAVRL FIEWLMNTK SEQ ID NO: 8 CA-SQGTFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO: 9 CA-GQGTFTSDYSRYLDEEAVXL FIEWLMNTKRNRNNIASEQ ID NO: 10 CA-SQGTFTSDYSRQMEEEAVRL FIEWLMNGGPSSGAPPPSK SEQ ID NO: 11CA-GEGTFTSDLSRQMEEEAVRL FIEWAAHSQGTFTSDYSRYLDK SEQ ID NO: 12CA-SQGTFTSDYSRYLDGGGHGE GTFTSDLSKQMEEEAVK SEQ ID NO: 13CA-SQGTFTSDYSRYLDXEAVXL FIEWLMNTK SEQ ID NO: 14 CA-GQGTFTSDYSRYLDEEAVXLFIXWLMNTKRNRNNIA SEQ ID NO: 15 CA-GQGTFTSDYSRYLDEEAVRL FIXWLMNTKRNRNNIASEQ ID NO: 16 CA-SQGTFTSDLSRQLEGGGHSQ GTFTSDLSRQLEK SEQ ID NO: 17CA-SQGTFTSDYSRYLDEEAVRL FIEWIRNTKRNRNNIA SEQ ID NO: 18CA-SQGTFTSDYSRYLDEEAVRL FIEWIRNGGPSSGAPPPSK SEQ ID NO: 19CA-SQGTFTSDYSRYLDEEAVKL FIEWIRNTKRNRNNIA SEQ ID NO: 20CA-SQGTFTSDYSRYLDEEAVKL FIEWIRNGGPSSGAPPPSK SEQ ID NO: 21CA-SQGTFTSDYSRQLEEEAVRL FIEWVRNTKRNRNNIA SEQ ID NO: 22DA-SQGTFTSDYSKYLDEKRAKE FVQWLMNTK SEQ ID NO: 23 HAibQGTFTSDYSKYLDEKRAKEFVCWLMNT SEQ ID NO: 24 HAibQGTFTSDYSKYLDEKRAKE FVQWLMNTC SEQ ID NO: 25HAibQGTFTSDYSKYLDEKRAKE FVQWLMNTC SEQ ID NO: 26 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO: 27 HAibQGTFTSDYSKYLDEQAAKE FICWLMNT SEQ ID NO: 28HAibQGTFTSDYSKYLDEKRAKE FVQWLMNT SEQ ID NO: 29 H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO: 30 CA-SQGTFTSDYSKYLDSRRAQD FVQWLMNTKRNRNNIASEQ ID NO: 31 CA-(d)SQGTFTSDYSKYLDSRR AQDFVQWLMNTKRNRNNIA SEQ ID NO: 32CA-AibQGTFTSDYSKYLDEKRA KEFVQWLMNTC SEQ ID NO: 33HAibQGTFTSDYAKYLDEKRAKE FVQWLMNTC SEQ ID NO: 34 YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC

In Table 1 above, the amino acids indicated by the bold letters meanring formation, and the amino acids indicated by X meanalpha-methyl-glutamic acid that is a non-native amino acid. In addition,CA indicates 4-imidazoacetyl, DA indicates desamino-histidyl, Aibindicates aminosiobutyric acid, and (d)S indicates d-serine.

Example 2-2 Measurement of In Vitro Activities of OxyntomodulinDerivatives

In order to measure the effects of anti-obesity peptides, the in vitroactivities of cells were measured using the transformants prepared inExamples 1-1 and 1-2.

Each of the transformants was transformed so as to express each of humanGLP-1 receptor and glucagon receptor genes in CHO (Chinese hamsterovary) and was suitable for measuring the activities of GLP-1 andglucagon. Thus, the activity of each of the oxyntomodulin derivativeswas measured using each of the transformants.

Specifically, each of the transformants was subcultured twice or threetimes a week, and the cells were dispensed into each well of a 96-wellplate at a density of 1×10⁵ cells/well and cultured for 24 hours.

The cultured cells were washed with KRB buffer, suspended in 40 ml of 1mM IBMX-containing KRB buffer, and then allowed to stand at roomtemperature for 5 minutes. Each of oxyntomodulin and the oxyntomodulinderivatives (SEQ ID NOs: 2-6, 8, 10-13, 17, 18, 23-25, 27, 28 and 32-34)was serially diluted by five-fold from 1000 nM to 0.02 nM, and 40 ml ofeach of the dilutions was added to the cells, which were then incubatedin a CO₂ incubator at 37° C. for 1 hour. Then, 20 ml of cell lysisbuffer was added to lyse the cells, and the concentration of cAMP ineach of the cell lysates was measured using a cAMP assay kit (MolecularDevice, USA). From the results of the measurement, EC₅₀ values werecalculated and compared with each other (Table2).

Table 2

TABLE 2 Comparison of in vitro activities of GLP-1 receptor and glucagonreceptor between oxyntomodulin derivatives EC₅₀(nM) SEQ ID NOsCHO/GLP-1R CHO/GCGR SEQ ID NO: 1  50-210 10-43  SEQ ID NO: 2 51.8 12.8SEQ ID NO: 3 >1,000 637.7 SEQ ID NO: 4 5.5 >1,000 SEQ ID NO: 55.9 >1,000 SEQ ID NO: 6 500.1 >1,000 SEQ ID NO: 8 419.6 >1,000 SEQ IDNO: 10 >1,000 >1,000 SEQ ID NO: 11 >1,000 >1,000 SEQ ID NO:12 >1,000 >1,000 SEQ ID NO: 13 >1,000 >1,000 SEQ ID NO: 17 97.9 >1,000SEQ ID NO: 18 96.3 >1,000 SEQ ID NO: 23 2.46 5.8 SEQ ID NO: 24 1.43 6.95SEQ ID NO: 25 1.9 1.3 SEQ ID NO: 27 2.8-5.5 3.1-5.6 SEQ ID NO: 28 3.10.3 SEQ ID NO: 32 41.3 17.7 SEQ ID NO: 33 2.2 80.2 SEQ ID NO: 34 12.51.04

As can be seen in Table 2 above, the oxyntomodulin derivatives showedexcellent in vitro GLP-1 and glucagon activities compared to theoxyntomodulin of SEQ ID NO:1.

Oxyntomodulin is known to have the effect of treating hyperlipidemia,fatty liver disease or arteriosclerosis by activating GLP-1 receptor andglucagon receptor. The oxyntomodulin derivatives according to thepresent invention have an excellent activity to activate GLP-1 receptorand glucagon receptor compared to native oxyntomodulin, and thus can beused to treat hyperlipidemia and the fatty liver disease andarteriosclerosis related to hyperlipidemia, in place of nativeoxyntomodulin.

Example 3 Preparation of a Conjugate Comprising an OxyntomodulinDerivative (SEO ID NO: 23) with Immunoglobulin Fc (ImmunoglobulinFc-Conjugated Oxyntomodulin Derivative 23)

In order to pegylate a cysteine residue at position 24 of anoxyntomodulin derivative of SEQ ID NO: 24 with MAL-10K-ALD PEG (NOF.,Japan), the oxyntomodulin derivative (SEQ ID NO: 23) and MAL-10K-ALD PEGwere allowed to react with each other at molar ratio of 1:3 at a proteinconcentration of 3 mg/ml at room temperature for 3 hours. The reactionwas performed in 50 mM Tris buffer (pH 8.0) containing 1M guanidine.After completion of the reaction, the reaction solution was purifiedusing SOURCE S under the following conditions, thereby obtaining anoxyntomodulin mono-pegylated into the cysteine: column: SOURCE S, flowrate: 2.0 ml/min, gradient: A 0→100% 50 min B (A: 20 mM Na-citrate, pH3.0+45% ethanol, B: A+1M KCl)).

Then, the purified mono-pegylated oxyntomodulin derivative (SEQ ID NO:23) and immunoglobulin Fc were allowed to react with each other at amolar ratio of 1:5 at a protein concentration of 20 mg/ml at 4° C. for16 hours. The reaction was performed in 100 mM potassium phosphatebuffer (pH 6.0) containing 20 mM SCB as a reducing agent. Aftercompletion of the reaction, the reaction solution was purified under thefollowing conditions, thereby obtaining a conjugate comprising theoxyntomodulin derivative (SEQ ID NO: 23) and immunoglobulin: column:SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A 0→4% 1 min, B→20% 80 minB (A: 20 mM Tris-HCl, pH 7.5, B: A+1M NaCl)); source ISO column: SOURCEISO, flow rate: 2.0 ml/min, gradient: B 0→100% 100 min A, (A: 20 mMTris-HCl, pH 7.5, B: A+1.1M AS).

Example 4 Preparation of a Conjugate Comprising an OxyntomodulinDerivative (SEQ ID NO: 25) with Immunoglobulin Fc (ImmunoglobulinFc-Conjugated Oxyntomodulin Derivative 25)

In order to pegylate a cysteine residue at position 30 of anoxyntomodulin derivative of SEQ ID NO: 25 with MAL-10K-ALD PEG, theoxyntomodulin derivative (SEQ ID NO: 25) and MAL-10K-ALD PEG wereallowed to react with each other at molar ratio of 1:3 at a proteinconcentration of 3 mg/ml at room temperature for 3 hours. The reactionwas performed in 50 mM Tris buffer (pH 8.0) containing 1M guanidine.After completion of the reaction, the reaction solution was purifiedusing SOURCE S under the following conditions, thereby obtaining anoxyntomodulin mono-pegylated into the cysteine: column: SOURCE S, flowrate: 2.0 ml/min, flow rate: A 0→100% 50 min B (A: 20 mM Na-citrate, pH3.0+45% ethanol, B: A+1M KCl).

Then, the purified mono-pegylated oxyntomodulin derivative (SEQ ID NO:25) and immunoglobulin Fc were allowed to react with each other at amolar ratio of 1:5 at a protein concentration of 20 mg/ml at 4° C. for16 hours. The reaction was performed in 100 mM potassium phosphatebuffer (pH 6.0) containing 20 mM SCB as a reducing agent. Aftercompletion of the reaction, the reaction solution was purified under thefollowing conditions, thereby obtaining a conjugate comprising theoxyntomodulin derivative (SEQ ID NO: 25) and immunoglobulin: SOURCE 15Qcolumn: SOURCE 15Q, flow rate: 2.0 ml/min, flow rate: A 0→4% 1 min B→20%80 min B (A: 20 mM Tris-HCl, pH 7.5, B: A+1M NaCl); and Source ISOcolumn: SOURCE ISO, flow rate: 2.0 ml/min, flow rate: B 0→100% 100 min A(A: 20 mM Tris-HCl, pH 7.5, B: A+1.1M AS).

Example 5 Effect of Long-Acting Oxyntomodulin on Reduction in Lipid inHyperlipidemia Model Hamsters Example 5-1 Grouping of Test Animals

8 week-old male hamsters (Golden Syrian hamsters, 120-130 g) werepurchased from Vital River China. It is known that hamsters show bloodlipid profiles similar to humans, unlike other rodents, and aresensitive to high-fat diets.

The animals were allowed access to a sterilized high-fat diet (Purina5001 containing 11.5% maize oil, 11.5% coconut oil, 0.5% cholesterol,and 0.25% deoxycholate; Dyets, Bethlehem, Pa.) or a standard rodent diet(low fat, 2018; Harlan Teklad, Madison, Wis.). A normal diet group wasallowed access to filtered and UV-sterilized tap water, and a high-fatdiet group was allowed access to water containing 10% fructose. Theanimals were kept in a breeding chamber satisfying GLP standards under a12-hr light/12-hr dark cycle (lighting: am 6 to pm 6), and all theexperimental procedures were performed according to the standardguideline for animal experiments. Drug administration was started after3 weeks of hyperlipidemia induction, and the animals were divided intofour groups (n=6) as shown in Table 3 below.

Table 3

TABLE 3 Method of Groups Drugs administered administration Normal groupVehicle (DPBS) Administered subcuta- Hyperlipidemia- Vehicle (DPBS)neously once a week induced group 3.25 nmol/kg of SEQ ID NO: 25-Fcconjugate 8.96 nmo/kg of SEQ ID NO: 23-Fc conjugate

Specifically, group 1 (normal group) was fed with a normal feed andadministered subcutaneously with 5 ml/kg of Dulbecco's phosphatebuffered saline (DPBS, Sigma) once or more a week.

Group 2 (hyperlipidemia-induced group) was fed with a high-fat diet toinduce hyperlipidemia, and then administered subcutaneously with 5 ml/kgof Dulbecco's phosphate buffered saline (DPBS, Sigma) once or more aweek.

Group 3 (hyperlipidemia-induced group+group administered with 3.25nmol/kg of SEQ ID NO: 25-Fc conjugate) was fed with a high-fat diet toinduce hyperlipidemia, and then administered with 3.25 nmol/kg of theSEQ ID NO: 25-Fc conjugate (prepared in Example 4) once a week at aninjection dose of 5 ml/kg.

Group 4 (hyperlipidemia-induced group+group administered with 8.96nmol/kg of SEQ ID NO: 23-Fc conjugate) was fed with a high-fat diet toinduce hyperlipidemia, and then administered with 8.96 nmol/kg of theSEQ ID NO: 23-Fc conjugate (prepared in Example 3) once a week at aninjection dose of 5 ml/kg.

Saline or the drug was administered into each group (n=6) for 2 weeks,and then the effects thereof on a reduction in lipid levels wereanalyzed.

Example 5-2 Analysis of Effect of Long-Acting Oxyntomodulin DerivativeConjugate on Reduction in Lipid Levels

In order to examine the effect of the long-acting oxyntomodulinderivative conjugate on a reduction in lipid levels in hamsters, thefollowing experiment was performed.

Blood was collected from the hamsters which were administered or notadministered with the long-acting oxyntomodulin derivative as describedin Example 5-1, and the lipid levels of the blood were analyzed usingHITACHI 7020. The results of the analysis are shown in FIGS. 1 to 5.

FIGS. 1 to 5 show the change in blood triglyceride levels (FIG. 1), thechange in blood total cholesterol levels (FIG. 2), the change inLDL-cholesterol levels (FIG. 3), blood HDL-cholesterol levels (FIG. 4),and the change in the blood HDL/LDL-cholesterol ratio (FIG. 5). Theobtained results were statistically processed, and the mean values andthe standard deviations of the mean values were calculated. In theverification of significance between the groups (n=6), data werestatistically processed using Dunnett's test of one-way ANOVA, and avalue of p<0.05 was considered statistically significant.

Specifically, in the results of measurement of blood triglyceridelevels, it was seen that, in the case of hamsters fed with a high-fatdiet, the triglyceride levels were significantly increased, but when thelong-acting oxyntomodulin derivative (SEQ ID NO: 25-Fc conjugate or SEQID NO: 23-Fc conjugate) was administered into the hamsters, thetriglyceride levels were significantly decreased (FIG. 1).

In the results of measurement of blood total cholesterol levels, it wasseen that, in the case of hamsters fed with a high-fat diet, the bloodtotal cholesterol levels were significantly increased, but when thelong-acting oxyntomodulin derivative (SEQ ID NO: 25-Fc conjugate or SEQID NO: 23-Fc conjugate) was administered into the hamsters, the bloodtotal cholesterol levels were significantly decreased (FIG. 2).

In the results of measurement of blood LDL-cholesterol levels, it wasseen that, in the case of hamsters fed with a high-fat diet, the bloodLDL-cholesterol levels were significantly increased, but when thelong-acting oxyntomodulin derivative (SEQ ID NO: 25-Fc conjugate or SEQID NO: 23-Fc conjugate) was administered into the hamsters, the bloodLDL-cholesterol cholesterol levels were significantly decreased (FIG.3).

In the results of measurement of blood HDL-cholesterol levels, the groupadministered with the SEQ ID NO: 25-Fc conjugate or the SEQ ID NO: 23-Fcconjugate showed a significant increase in the blood HDL-cholesterollevels compared to the high-fat diet hamster group (FIG. 4).

In the results of measurement of blood HDL/LDL-cholesterol levels, thegroup administered with the SEQ ID NO: 25-Fc conjugate or the SEQ ID NO:23-Fc conjugate showed a significant increase in the bloodHDL/LDL-cholesterol ratio compared to the high-fat diet hamster group(FIG. 5).

From the above results, it could be seen that the inventiveoxyntomodulin derivative conjugate comprising the immunoglobulin Fcregion covalently linked to the oxyntomodulin derivative by PEG preventsthe accumulation of blood triglyceride and low-density (LDL)cholesterol, and thus can be effectively used for the treatment ofhyperlipidemia or related fatty liver disease or arteriosclerosis.

Example 6 Analysis of Effects of Known Long-Acting GLP-1 Analog andLong-Acting Oxyntomodulin Derivative Conjugate

VICTOZA® is a long-acting glucagon-like peptide-1, GLP-1 analog which iscurrently marketed as an agent for treating diabetes and is known tohave the effects of treating obesity and increasing HDL cholesterollevels.

The effect of reducing lipid levels was compared between theoxyntomodulin derivative conjugate and known VICTOZA®.

As described in Example 5, hamsters were divided into a normal hamstergroup and hamster groups fed with a high-fat diet. The normal hamstergroup was administered subcutaneously with 5 ml/kg of DPBS once or morea week. The hamster groups fed with high-fat diet were divided into agroup administered subcutaneously with 5 ml/kg of DPBS once or more aweek, a group administered subcutaneously with 35.5 nmol/kg of VICTOZA®once or more a week, a group administered subcutaneously with 3.25nmol/kg of the SEQ ID NO: 25-Fc conjugate, and a group administeredsubcutaneously with 8.96 nmol/kg of the SEQ ID NO:23-Fc conjugate, andthe blood lipid levels of the groups were analyzed.

As a result, it could be seen that administration of the inventivelong-acting oxyntomodulin derivative conjugate (SEQ ID NO: 25-Fcconjugate or SEQ ID NO: 23-Fc conjugate) showed a decrease in bloodtotal cholesterol levels (FIG. 6) and a decrease in bloodLDL-cholesterol level (FIG. 7) compared to administration of commercialVICTOZA®.

In addition, it could be seen that administration of the inventivelong-acting oxyntomodulin derivative conjugate (SEQ ID NO: 25-Fcconjugate or SEQ ID NO: 23-Fc conjugate) showed increases in bloodHDL-cholesterol levels and the HDL/LDL-cholesterol ratio compared toadministration of VICTOZA®(FIGS. 8 and 9). Particularly, the long-actingSEQ ID NO: 25-Fc conjugate showed significant increases in bloodHDL-cholesterol levels and the HDL/LDL-cholesterol ratio compared toVICTOZA®.

In addition, administration of the inventive long-acting oxyntomodulinderivative conjugate (SEQ ID NO: 25-Fc conjugate or SEQ ID NO: 23-Fcconjugate) showed a decrease in blood triglyceride levels compared toadministration of VICTOZA®.

From the above results, it can be seen that the long-actingoxyntomodulin derivative conjugate of the present invention exhibits alipid-lowering effect that is equal to or higher than that of knownVICTOZA®, and thus the conjugate can be effectively used as an agent fortreating hyperlipidemia, fatty liver disease or arteriosclerosis.

1. A composition for preventing or treating hyperlipidemia, fatty liverdisease or arteriosclerosis, comprising an oxyntomodulin derivative asan active ingredient.
 2. The composition according to claim 1, whereinthe oxyntomodulin derivative has an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2 to
 34. 3. The compositionaccording to claim 1, wherein the oxyntomodulin derivative is in theform of a conjugate linked to one selected from the group consisting ofan immunoglobulin fragment, an antibody, elastin, albumin andfibronectin.
 4. The composition according to claim 3, wherein theconjugate is a conjugate in which the oxyntomodulin derivative havingthe amino acid sequence selected from the group consisting of SEQ IDNOs: 2 to 34 is linked to an immunoglobulin Fc region via a non-peptidylpolymer.
 5. The composition according to claim 4, wherein thenon-peptidyl polymer is selected from the group consisting ofpolyethylene glycol, polypropylene glycol, an ethylene glycol/propyleneglycol copolymer, polyoxyethylated polyol, polyvinyl alcohol,polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid),PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronicacid, and combinations thereof.
 6. The composition according to claim 4,wherein one end of the non-peptidyl polymer and the other are linked toan amine group or a thiol group of the immunoglobulin Fc region and theoxyntomodulin derivative, respectively.
 7. The composition according toclaim 1, further comprising a pharmaceutical agent showing preventive ortherapeutic effects against hyperlipidemia, fatty liver disease orarteriosclerosis.
 8. The composition according to claim 1, wherein thefatty liver disease is non-alcoholic fatty liver disease, alcoholicfatty liver disease, nutritional fatty liver disease, starvation fattyliver disease, obesity fatty liver disease, diabetic fatty liver diseaseor steatohepatitis.
 9. The composition according to claim 8, wherein thenon-alcoholic fatty liver disease results from hyperlipidemia, diabetesor obesity.
 10. The composition according to claim 8, wherein thenon-alcoholic fatty liver disease is selected from the group consistingof simple steatosis, non-alcoholic steatohepatitis, liver fibrosis andliver cirrhosis.
 11. A method for preventing or treating hyperlipidemia,fatty liver disease or arteriosclerosis, comprising a step ofadministering an oxyntomodulin derivative to a subject.
 12. Use of anoxyntomodulin derivative in preparation of a medicament for preventingor treating hyperlipidemia, fatty liver disease or arteriosclerosis.